A Correlative Method for Imaging Identical Regions of Samples by Micro-CT, Light Microscopy, and Electron Microscopy: Imaging Adipose Tissue in a Model System
A Correlative Method for Imaging Identical Regions of Samples by Micro-CT, Light Microscopy, and Electron Microscopy: Imaging Adipose Tissue in a Model System
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DOI：
10.1369/0022155412473757
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发表时间：
2013-04-01
影响因子：
3.2
通讯作者：
Keene, Douglas R.
中科院分区：
文献类型：
--
作者：
Sengle, Gerhard;Tufa, Sara F.;Keene, Douglas R.
我们提出了一种方法，该方法通过非侵入性微计算机x射线断层扫描（micro-CT）在完整生物体内的精确区域进行三维空间映射，然后通过光学显微镜（LM）和透射电子显微镜（TEM）进一步评估。组织被准备为TEM，包括锇固定，由于其强x射线衰减，在微ct中赋予软组织对比。因此，该方法可以应用于嵌入的、存档的TEM样品。在三维体积内从感兴趣区域（ROI）选择二维（2-D）投影后，将环氧树脂嵌入的样品定向进行显微切开术，使切面与micro-CT投影对齐。通过将LM图像与二维微ct投影叠加来验证配准。在微ct中分辨率较差的结构可以通过观察下一个系列超薄切片在TEM分辨率下进行评估，从而通过所有三种成像技术获得相同的ROI。我们比较了含有脂质改变突变的小鼠前肢内的白色脂肪组织与它们的同窝对照。我们证明，单个锇染色的脂滴小至15 μ m，相隔仅35 μ m，可以在微型ct中识别为单独的实体，验证了这是一种高分辨率，非破坏性的脂肪含量评估技术。
We present a method in which a precise region of interest within an intact organism is spatially mapped in three dimensions by non-invasive micro-computed X-ray tomography (micro-CT), then further evaluated by light microscopy (LM) and transmission electron microscopy (TEM). Tissues are prepared as if for TEM including osmium fixation, which imparts soft tissue contrast in the micro-CT due to its strong X-ray attenuation. This method may therefore be applied to embedded, archived TEM samples. Upon selection of a two-dimensional (2-D) projection from a region of interest (ROI) within the three-dimensional volume, the epoxy-embedded sample is oriented for microtomy so that the sectioning plane is aligned with the micro-CT projection. Registration is verified by overlaying LM images with 2-D micro-CT projections. Structures that are poorly resolved in the micro-CT may be evaluated at TEM resolution by observing the next serial ultrathin section, thereby accessing the same ROI by all three imaging techniques. We compare white adipose tissue within the forelimbs of mice harboring a lipid-altering mutation with their littermate controls. We demonstrate that individual osmium-stained lipid droplets as small as 15 mu m and separated by as little as 35 mu m may be discerned as separate entities in the micro-CT, validating this to be a high-resolution, non-destructive technique for evaluation of fat content.